Human papillomavirus 44 nucleic acid hybridization probes and methods for employing the same

ABSTRACT

Nucleic acid hybridization probes for human papillomavirus types and particularly human papillomavirus type 44; and methods for employing the same.

FIELD OF THE INVENTION

The present invention relates to nucleic acid hybridization probes forhuman papillomavirus types and particularly for human papillomavirustype 44 (hereinafter "HPV 44"); and methods for employing the same.

BACKGROUND OF THE INVENTION (A) Human Papillomavirus Types

Human Papillomavirus (hereinafter "HPV") are recognized as a cause ofvarious epithelial lesions such as warts, condylomas and dysplasias (seeGissman, L., Cancer Surv., 3:161 (1984): Pfister, H. Biochem.Pharmacol., 99:111 (1983); Durst, M. et al, Proc. Natl. Acad. Sci. USA,80:3812 (1983) and Boshart, M. et al, EMBO J., 3:1151 (1984)).Dysplasias of the cervix (also known as cervical intraepithelialneoplasia (CIN)) are believed to be early events in the progression tocervical cancer; the progression proceeding from mild dysplasia (CIN I),to moderate dysplasia (CIN II), to severe dysplasia, to carcinoma insitu (collectively CIN III), to invasive cancer.

Studies examining the association of HPV type with dysplasias of thecervix and cancer of the cervix have shown that HPV types 6, 11, 16, 18,31 and 33 are associated with a high percentage of genital lesions (seeGissman, L., Cancer Surv., 3:161 (1984); Pfister, H., Biochem.Pharmacol., 99:111 (1983); Durst, M. et al, Proc. Natl. Acad. Sci. USA,80:3812 (1983); Boshart, M. et al, EMBO J., 3:1151 (1984); de Villiers,E. -M. et al, J. Virol., 40:932 (1981); Gissman, L. et al, J. Virol.44:393 (1982); Lorincz, A.T. et al J. Virol., 58:225 (1986) andBeaudenon, S., Nature, 321:246 (1986)).

HPVs are grouped into types based on the similarity of their DNAsequence. Two HPVs are taxonomically classified as being of the sametype if their DNAs cross-hybridize to greater than 50%, as measured byhybridization in solution under moderately stringent hybridizationconditions, which are defined as approximately 25° C. below the meltingtemperature of a perfectly base-paired double-stranded DNA (convenientlywritten as T_(m) -25° C.), followed by chromatography on hydroxyapatiteto separate double-stranded DNA from single-stranded DNA (see Coggin,J.R. et al, Cancer Res., 39:545 (1979)). The melting temperature (T_(m))of a perfectly base-paired double-stranded DNA can be accuratelypredicted using the following well established formula:

    T.sub.m 32 16.6×log[Na.sup.+ 1+0.41×%G:C+81.5-0.72×(%)(v/v) formanide

The above formula provides a convenient means to set a reference pointfor determining non-stringent and stringent hybridization conditions forvarious DNAs in solutions having varying salt and formamideconcentrations without the need for empirically measuring the T_(m) foreach individual DNA in each hybridization condition.

If less than 50% of the respective HPV DNAs are able to cross-hybridizein solution under moderately stringent conditions to form fully orpartially double-stranded structures, as measured and defined by theability to bind to hydroxyapatite, then the HPV DNAs are notsufficiently related to be taxonomically classified as being of the sametype. A cut-off of 50% cross-hybridization using this method is employedas the consensus criterion for the assignment of novel HPV types fornomenclature purposes. This method for measuring the degree ofcross-hybridization between HPV DNAs has been historically adopted asthe method to be used to determine whether two HPV DNAs representdifferent isolates of a common type or represent isolates of differenttypes. The use of this criterion pre-dates the establishment of clinicalcriterion for determining and defining HPV types. As discussed in moredetail below, the clinical criterion for determining and defining HPVtypes is based upon the epidemiological distribution of HPV types amonggenital lesions.

The above-described method of measuring the degree ofcross-hybridization is based on an assessment of the extent of formationof fully or partially double-stranded DNA molecules after thehybridization reaction. However, it should be noted that conversion of50% of the DNAs into fully or partially double-stranded DNA moleculesdoes not imply that the nucleotide sequences of the DNAs are 50%homologous.

As discussed above, HPVs can also be grouped into types based onclinical criterion. That is, it has been observed that HPV of differenttypes, as defined by the degree of cross-hybridization criteriondescribed above, show distinct epidemiological distributions amonggenital lesions of different severities and among different geographicpopulations.

For example, HPV 6 and HPV 11 are principally associated with benignlesions such as exophytic condylomas and to a lesser extent with flatcondylomas (see Gissman, L. et al, Proc. Natl. Acad. Sci., USA, 80:560(1983)). HPV 6 and HPV 11 are also detected in certain rare types ofmalignant epithelial tumors (see Zachow, K.R. et al, Nature, 300:771(1982) and Rando, R.F., J. Virol, 57:353 (1986)). In contrast, HPV 16,HPV 18, HPV 31 and HPV 33 are detected with varying degrees of frequencyin cervical and other anogenital cancers as well as their precursorlesions (see Durst, M. et al, Proc. Natl. Acad. Sci., USA, 80:3812(1983), Boshart, M. et al, Embo. J. 3:115 (1984), Lorincz, A.T. et al,J. Virol., 58:225 (1986) and Beaudenon, S., Nature, 321:246 (1986)).This distribution of HPV 16, HPV 18, HPV 31 and HPV 33 is believed toreflect a greater risk of, or a more rapid progression to, cervicalcancer arising from genital lesions infected with HPV 16, HPV 18, HPV 31and HPV 33 as compared to lesions infected with HPV 6 and HPV 11. As aresult, the determination of HPV types has clinical-diagnostic value,i.e., such is an important factor in the assessment of risk of cancerdevelopment in patients who exhibit evidence of HPV infection. Based onthe assessed risk of cancer development, appropriate therapeutictreatments can be selected.

In addition, HPV 16 is more prevalent in Europe than in Africa (Durst,M. et al, Proc. Natl. Acad. Sci., USA, 80:3812 (1983)), whereas HPV 18is more prevalent in Africa than in Europe (Boshart, M. et al, EMBO, J.,3:1115 (1984)).

Accordingly, within the context of the present invention, two HPVs areconsidered to be of the same type if either (1) they meet the criterionfor the degree of cross-hybridization discussed above or (2) if theyshow substantially the same epidemiological distribution ofcross-hybridization among genital lesions and they both cross-hybridizewith the same genital lesions which comprise the epidemiologicaldistribution.

It has been found that a significant percentage of cervical cancer andgenital lesions which have the potential to progress to cervical cancercontain "new" HPV types which do not correspond to any of the known HPVtypes. Thus, in light of the known association of specific HPV typeswith genital lesions which have a high risk of progression to cervicalcancer, the ability to detect and group these "new" HPV types allows therisk of cervical cancer associated with these "new" HPV types to beascertained in patients who exhibit evidence of HPV infection and whomay be infected with these "new" HPV types.

(B) Cloning of HPV Types

In spite of long standing efforts in the art, it has not been possibleto propagate HPV in cell culture in vitro. However, recombinant DNAcloning techniques have made it possible to isolate and purify the DNAof many HPV types such as HPV Types 6, 11, 16, 18, 31 and 33 (see Durst,M. et al, Proc. Natl. Acad. Sci. USA, 80:3812 (1983)); Boshart, M. etal, EMBO J., 3:1151 (1984); de Villiers, E. -M. et al, J. Virol., 40:932(1981); Gissmann, L. et al, J. Virol., 44:393 (1982); Lorincz, A.T. etal J. Virol., 58:225 (1986) and Beaudenon, S., Nature, 321:246 (1986)).Most of the knowledge regarding HPVs has been derived from the study ofthe DNA sequence in such recombinant DNAs and the use of these DNAs toprepare nucleic acid hybridization probes for detection of HPV in tissuesamples.

(C) Hybridization Probes

As discussed above, HPV DNA has been employed as hybridization probes todifferentiate HPV types. Two HPV DNAs of different types can be readilydistinguished by hybridization under stringent hybridization conditions,which are defined as approximately 10° C. below the melting temperatureof a perfectly based-paired double-stranded DNA hybrid (convenientlywritten as T_(m) -10° C.), using such hybridization probes. Similarly,an HPV DNA of one type can be readily distinguished from an HPV RNA ofanother type by hybridization under stringent hybridization conditionswhich are defined as approximately 10° C. below the melting temperatureof a perfectly based-paired double-stranded DNA-RNA hybrid (convenientlywritten as T_(m) -10° C.), using such hybridization probes. Further, twoHPV RNAs of different types can be readily distinguished byhybridization under stringent hybridization conditions, which aredefined as approximately 10° C. below the melting temperature of aperfectly based-paired double-stranded RNA-RNA hybrid (convenientlywritten as T_(m) -10° C.), using such hybridization probes. It should benoted that HPV DNAs or RNAs which are designated as different typesusing the above criterion, may in fact have as much as 80% of theirnucleotide sequences in common.

Furthermore, two HPV DNAs of different types are able to cross-hybridizeunder non-stringent hybridization conditions, which are defined asapproximately 35° C. or more below the melting temperature of aperfectly base-paired double-stranded DNA-DNA hybrid (convenientlywritten as T_(m) -35° C. or more), using such hybridization probes.Similarly, an HPV DNA of one type is able to cross-hybridize with an HPVRNA of another type by hybridization under non-stringent hybridizationconditions which are defined as approximately 35° C. or more below themelting temperature of a perfectly based-paired double-stranded DNA-RNAhybrid (conveniently written as T_(m) -35° C. or more), using suchhybridization probes. Further, two HPV RNAs of different types are ableto cross-hybridize under non-stringent hybridization conditions, whichare defined as approximately 35° C. or more below the meltingtemperature of a perfectly based-paired double-stranded RNA-RNA hybrid(conveniently written as T_(m) -35° C. or more), using suchhybridization probes (see Anderson, L.M. et al, Nucleic AcidHybridization, pages 73-111, Eds. B.D. Hames and S.J. Higgins, I.R.L.Press, Oxford, England and Washington, D.C., USA (1985)).

The melting temperatures of DNA-DNA, DNA-RNA and RNA-RNA hybrids of thesame nucleotide sequences may be different in various chemicalenvironments. The effect of various compounds on the relative meltingtemperatures of these various hybrids has been studied for severalagents. For example, it is well known that increasing the concentrationof formamide differentially destabilizes DNA-DNA hybrids more thanDNA-RNA hybrids so that at high concentrations of formamide, such as 80%(v/v), a DNA-RNA hybrid may have a significantly higher meltingtemperature than a DNA-DNA hybrid of the same nucleotide sequence.

As discussed above, the melting temperature of a DNA-DNA hybrid can bepredicted as described in Anderson, L.M. et al, Nucleic AcidHybridization, pages 73-111, Eds. B.D. Hames and S.J. Higgins, I.R.L.Press, Oxford, England and Washington, D.C., USA (1985)). Further, themelting temperature of a DNA-DNA hybrid can be empirically determined asdescribed in Howley, P. et al, J. Biochem., 254:4876 (1979). The meltingtemperature of a DNA-RNA hybrid and a RNA-RNA hybrid can also bedetermined by means well known in the art.

Thus, it is possible to test a tissue sample for the presence of HPV DNAor RNA in general and/or a particular HPV DNA or RNA type by nucleicacid hybridization depending upon what conditions, i.e., stringent ornon-stringent, are employed for hybridization.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to ascertain whethercervical cancers and genital lesions, which have the potential toprogress to cervical cancer, contain a "new" HPV type(s) and if so, toclone the hypothesized "new" HPV type(s).

Another object of the present invention is to provide nucleic acidhybridization probes which are specific for HPV types in general and forthe "new" HPV type(s) in particular.

Still another object of the present invention is to provide a method fordetecting HPV DNA or RNA in general and the "new" HPV type(s) DNA or RNAin particular, in an unknown sample of DNA or RNA, particularly anunknown sample of DNA or RNA derived from a genital lesion so as todetermine the risk of cervical cancer development.

These and other objects of the present invention will be apparent fromthe detailed description of the invention provided hereinafter.

It has been found in the present invention that the "new" HPV typecloned in the present invention is a novel HPV type, designated HPV 44.

Thus, in one embodiment, the above-described objects of the presentinvention have been met by a recombinant DNA of HPV 44 comprising acloning vector and substantially all of HPV 44 DNA or fragments thereof.

In other embodiments, the above-described objects of the presentinvention have been met by essentially pure HPV 44 DNA or fragmentsthereof or HPV 44 RNA or fragments thereof, or mixtures thereof, and bynucleic acid hybridization probes for HPV DNA or RNA in general and HPV44 DNA or RNA in particular which comprise the above-described DNAs orRNAs which have been labelled with a detectable marker.

In still another embodiment, the above-described objects of the presentinvnetion have been met by a method for detecting HPV DNA or RNAcomprising:

(1) carrying out hybridization, under non-stringent conditions, with

(a) a member selected from the group consisting of

(i) HPV 44 DNA or fragments thereof labelled with a marker, and

(ii) HPV 44 RNA or fragments thereof labelled with a marker;

(b) an unknown sample of DNA or RNA, and

(2) assaying for the presence of cross-hybridization so as to detect HPVDNA or RNA in said sample.

In a further embodiment, the above-described objects of the presentinvention have been met by a method for detecting HPV 44 DNA or RNAcomprising:

(1) carrying out hybridization, under stringent conditions, with

(a) a member selected from the group consisting of

(i) HPV 44 DNA or fragments thereof labelled with a marker, and

(ii) HPV 44 RNA or fragments thereof labelled with a marker;

(b) an unknown sample of DNA or RNA, and

(2) assaying for the presence of cross-hybridization so as to detect HPV44 DNA or RNA in said sample.

In a still further embodiment, the above-described objects of thepresent invention have been met by a method for detecting HPV 44 DNA orRNA comprising:

(1) carrying out hybridization, under stringent conditions, with

(a) a first fraction of DNA or RNA derived from each genital lesion of asampling of genital lesions, which sampling shows an epidemiologicalprogression to cervical cancer, and

(b) a member selected from the group consisting of

(i) HPV 44 DNA or fragments thereof labelled with a marker, and

(ii) HPV 44 RNA or fragments thereof labelled with a marker;

(2) carrying out hybridization, under stringent conditions, with

(a) a second fraction of DNA or RNA derived from each genital lesion ofsaid sampling of genital lesions, and

(b) an unknown sample of DNA derived from a genital lesion labelled witha marker;

(3) comparing the epidemiological distribution of cross-hybridizationobtained in Step (1) with that obtained in Step (2) and thecross-hybridization of the DNA of each lesion which comprises saidepidemiological distribution so as to detect HPV 44 DNA or RNA in saidsample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the restriction nuclease map of HPV 44 DNA.

FIG. 2 shows regions of partial homology between HPV 6 and HPV 44 DNA asdetermined by nucleic acid hybridization under non-stringenthybridization conditions. The arrows connect regions which exhibithomology. The dotted arrows indicate regions which have only weakhomology. Each of the maps in FIG. 2 is arranged so that the ends of thelinear map correspond to the relative position of the HpaI site of HPV6. The positions of the open reading frames deduced for HPV 6 are shownabove the homology map.

FIG. 3 graphically illustrates the distribution of HPV types in variouslesions based on the data in Table 1.

DETAILED DESCRIPTION OF THE INVENTION

A previously unknown HPV type has been found in the present invention,and designated HPV 44. HPV 44 has been cloned for the first tie in thepresent invention, thus enabling the preparation of nucleic acidhybridization probes for the detection of HPV DNA or RNA in general andHPV 44 DNA or RNA in particular in an unknown sample of DNA or RNA,particularly an unknown sample of DNA or RNA derived from genitallesions.

HPV 44 was isolated and cloned from a vulvar condyloma biopsy obtainedfrom Michigan.

The specific cloning vector employed in the example provided herein toinitially clone HPV 44 to prepare HPV 44 clone was λ L47. Thereafter,the HPV 44 DNA from HPV 44 clone 1 was subcloned in pT713 (GIBCO/BRL,Gaithersburg, MD) to prepare HPV 44 clone 2. HPV 44 clone 2 has beendeposited at the American Type Culture Collection under ATCC No. 40353.

HPV 44 DNA in its entirety can be excised from HPV 44 clone 2 usingBamHI restriction endonuclease and subcloned in any well knownprocaryotic and eucaryotic cloning vectors. The particular cloningvector employed for subcloning HPV 44 is not critical and can be anyknown procaryotic cloning vector such as pUC11, λ derived vectors suchas λ charon or M13 derived bacteriophages (see Maniatis, T. et al,Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, New York (1982) and Loenen, W.A.M. et al, Gene,20:249 (1980)) or any known eucaryotic cloning vector such as pZIP-NeoSV [X1] or pBKTK-1 (see Poueels, P.H. et al, Cloning Vectors: ALaboratory Manual, Elseiver, Amsterdam (1985)).

Fragments of HPV 44 DNA can similarly be excised from HPV 44 clone 2using other well known restriction endonucleases and cloned in theabove-described cloning vectors.

The cloning of HPV 44 DNA or fragments thereof allows for the relativelysimple production of large amounts of HPV 44 DNA or fragments thereoffor use in the preparation of nucleic acid hybridization probes for HPVDNA or RNA in general and HPV 44 DNA or RNA in particular.

In addition, HPV 44 DNA or fragments thereof can be subcloned in otherwell known cloning vectors to take advantage of special properties ofparticular cloning vectors which facilitate the synthesis, in vitro, ofRNA homologous to the HPV 44 DNA inserted into the cloning vector (seeManiatis, T. et al, Molecular cloning: A laboratory manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, New York (1982)). Examples ofthese cloning vectors include pT712 and pT713, each of which iscommercially available from GIBCO/BRL, Gaithersburg, MD. HPV 44 DNA orfragments thereof can be subcloned into these cloning vectors so thatthe HPV 44 DNA or fragments thereof can serve as an efficient templatefor phage encoded RNA polymerases, e.g., T7, T3 or SP6. Using suchcloning vectors and such RNA polymerases, HPV 44 RNA complementary toeither one of the strands of HPV 44 DNA or fragments thereof can besynthesized by in vitro transcription using methods well known in theart.

The specific bacterial or eucaryotic hosts for growing the cloningvectors containing HPV 44 DNA for fragments thereof will depend upon thecloning vector employed. For example, a typical host for growing HPV 44DNA cloned in λ L47 includes E. coli NM538 (Frischanf, A.M. et al, J.Mol. Biol., 170:827 (1983)). Other hosts such as E. coli HB101 (Boyer,H.W. et al, J. Mol. Biol., 41:459 (1969)) can be employed when usingpBR322 or pUC11 as the cloning vector. A typical host for growing HPV 44DNA cloned in pZIP-Neo SV [X1] is Monkey Cos cells while a typical hostfor growing HPV DNA cloned in pBKTK-1 would be any of a number of wellknown mammalian cell lines (see Poueels, P.H. et al, Cloning Vectors: ALaboratory Manual, Elseiver, Amsterdam (1985)).

The hybridization of the probes of the present invention to HPV DNA orRNA in general or to HPV 44 DNA or RNA in particular will depend uponthe hybridization conditions employed. That is, under non-stringenthybridization conditions, HPV 44 DNA or fragments thereof or HPV 44 RNAor fragments thereof can be employed as hybridization probes for HPV DNAor RNA in general. On the other hand, under stringent hybridizationconditions, HPV 44 DNA or fragments thereof or HPV 44 RNA or fragmentsthereof can be employed as hybridization probes for HPV 44 DNA or RNA inparticular.

As discussed above, the DNAs and RNAs of different types of HPV are ableto cross-hybridize under non-stringent hybridization conditions, i.e.,approximately 35° C. or more below the melting temperature of aperfectly base-paired double-stranded DNA having a base compositionequal to that of HPV DNAs or RNAs as a general group.

Furthermore, it is possible to test an unknown sample of DNA or RNA forthe presence of a particular HPV type and to identify that type bycarrying out hybridization under stringent hybridization conditions,i.e., approximately 10° C. below the melting temperature of a perfectlybase-paired double-stranded DNA having a base composition equal to thatof HPV DNAs or RNAs as a general group.

In the methods of the present invention, hybridization undernon-stringent conditions is carried out by first hybridizing undernon-stringent hybridization conditions followed by washing undernon-stringent hybridization conditions.

In addition, in the methods of the present invention, hybridizationunder stringent conditions is carried out by either first hybridizingunder non-stringent hybridization conditions followed by washing understringent hybridization conditions or by first hybridizing understringent hybridization conditions followed by washing under stringenthybridization conditions. In the first method, i.e., first hybridizingunder non-stringent hybridization conditions followed by washing understringent hybridization conditions, hybrids which form between DNAs orRNAs of different types are unstable but hybrids which form between DNAsor RNAs of the same type are stable.

To determine if an unknown sample of DNA or RNA is of the same ordifferent HPV type as the hybridization probe employed, hybridization ispreferably carried out under non-stringent hybridization conditionsfollowed by washing under non-stringent hybridization conditions. Afterassaying for the presence of hybrids, the detected hybrids are washedunder stringent hybridization conditions. In this method, the amount ofhybrids which remain after hybridizing under non-stringent hybridizationconditions are determined and compared with the amount of hybridspresent after washing under stringent hybridization conditions resultsin no or minimal reduction in the amount of hybrids formed, then thisindicates that the hybrids originally formed, i.e., the ones whichformed under non-stringent conditions, were between DNAs or RNAs of thesame type. Conversely, abolition of hybrids or a severe reduction of theamount of hybrids which remain after washing under stringenthybridization conditions indicates that the hybrids originally formed,i.e., ones which formed under non-stringent hybridization conditions,were between DNAs or RNAs of different types.

The ability of the HPV DNA or RNA to bind to the unknown sample of DNAor RNA under stringent hybridization conditions is indicative of a highdegree of nucleotide sequence hemology. On the other hand, the abilityof the HPV DNA or RNA to bind to the unknown sample of DNA or RNA onlyunder non-stringent hybridization conditions is indicative of a low orintermediate degree of nucleotide sequence homology. The exact degree ofnucleotide sequence homology can only be determined by directlysequencing the unknown DNA and comparing that with the known sequence ofthe HPV DNA.

In situations in which the detection of HPV DNA or RNA in general in anunknown sample of DNA or RNA, particularly in an unknown sample of DNAor RNA derived from a genital lesion, is being carried out, it is, as apractical matter, advantageous to utilize a hybridization probecomposition comprising a mixture of hybridization probes. Thesehybridization probes comprise probes with sequences representative ofall or most of the types suspected of being present in the unknownsample of DNA or RNA. A hybridization probe mixture of DNA or RNAsequences representative of HPV Types 6, 11, 16, 18, 31, 33 and 44 isparticularly advantageous when the unknown sample of DNA or RNA isderived from a genital lesion because these HPV types are most likely tobe found in genital lesions. Other known HPV types are seldom or neverfound in genital lesions. For example, HPV Types 1, 2 and 4 aregenerally found in other types of lesions, i.e., cutaneous warts (seeHeilman, C.A. et al, J. Virol., 360:395 (1980)) and thus a hybridizationprobe mixture of DNA or RNA sequences containing HPV Types 1, 2 and 4may be advantageous when the unknown sample of DNA or RNA is derivedfrom cutaneous warts but is not as useful when the unknown sample of DNAor RNA is derived from genital lesions.

Examples of sequences of HPV Types 6, 11, 16, 18, 31 and 33 which can beemployed in the hybridization probe mixture are described in Gissman,L., Cancer Surv., 3:161 (1984); Pfister, H., Biochem. Pharmacol., 99:111(1983); Durst, M. et al, Proc. Natl. Acad. Sci. USA, 80:3812 (1983);Boshart, M. et al, EMBO J., 3:1151 (1984); Lorincz, A.T. et al J.Virol., 58:225 (1986) and Beaudenon, S., Nature, 321:246 (1986).Further, examples of sequences of HPV Types 1, 2 and 4 which can beemployed in the hybridization probe mixture are well known in the art(see Heilman, C.A. et al, J. Virol., 360:395 (1980)). Thus, with thedisclosure herein as to HPV 44 and with the knowledge of one skilled inthe art as to HPV Types 6, 11, 16, 18, 31 and 33 and to other HPV typessuch as HPV Types 1, 2 and 4, hybridization probe mixtures can bereadily prepared.

In the hybridization probe mixtures, the particular percentage of DNAsor RNAs of each HPV type is not critical in the present invention.Generally, roughly equal molar amounts of DNAs or RNAs of each HPV typeare employed in the mixture.

Nucleic acid hybridization as a means of detecting and typing HPV can becarried out in solution as described above (see Loggins, J.R. et al,Cancer Res., 39:545 (1979)) or on a solid support (see Maniatis, T. etal, Molecular cloning: a laboratory manual, Cold Spring HarborLaboratory, Cold Spring Harbor, New York (1982) or in situ (see Brigati,D.J. et al, Virol., 126:32 (1983) and Beckmann, A.M. et al, J. Med.Virol., 16:265 (1985)).

Hybridization on a solid support can be carried out using a number ofdifferent procedures. One such procedure involves purifying all of theunknown DNAs or RNAs, immobilizing such to a solid support insingle-stranded form, followed by hybridization with labelled HPV 44 DNAor fragments thereof or labelled HPV 44 RNA or fragments thereof.

Alternatively, the purified unknown DNAs can be digested with one ormore restriction endonucleases and the resulting DNA fragments in thesamples can be separated electrophoretically. The DNA fragments can thenbe transferred to a solid support and hybridized with labelled HPV 44DNA or fragments thereof or labelled HPV 44 RNA or fragments thereof.

Hybridization in situ is performed on glass slides and the end result ofthe procedure is viewed through a microscope. In this procedure, the DNAor RNA is not purified from the cells but is left with all of the othercellular components.

HPV 44 RNA or fragments thereof are preferably used as nucleic acidhybridization probes for HPV DNA in general and HPV 44 DNA in particularwhen using crude extracts, particularly crude genital lesion extractsrather than purified DNA, e.g., from such genital lesions.

When employing HPV 44 RNA as a hybridization probe for detecting HPV 44DNA in an unknown sample of DNA, it is preferably that the DNA-RNAhybrids formed after first hybridizing under stringent hybridizationconditions, are treated with pancreatic RNaseA (about 20 mg/ml in 50 mMNaCl (pH 7.0)) at room temperature, followed by washing under stringenthybridization conditions.

The HPV 44 DNA or fragments thereof or HPV 44 RNA or fragments thereofare useful as nucleic acid hybridization probes for HPV DNA or RNA ingeneral and HPV 44 DNA or RNA in particular when labelled with aradioactive marker such as ³² P, ¹⁴ C, ³ H, ¹²⁵ ;I or ³⁵ S.

HPV 44 DNA or fragments thereof can be radioactively labelled, forexample, by "nick-translation" by well known means, as described in, forexample, Rigby, P.J.W. et al, J. Mol. Biol., 113-237 (1977) and by T4DNA polymerase replacement synthesis as described in, for example, Deen,K.C. et al, Anal. Biochem., 135:456 (1983)).

HPV 44 RNA or fragments thereof can be labelled with a radioactivemarker by in vitro transcription as described in, for example, Davanloo,P. et al, Proc. Natl. Acad. Sci., USA, 81:2035 (1984)). Since RNApolymerases can utilized labelled precursors, it is possible tosynthesize labelled RNA by this method so as to prepare HPV 44 RNAprobes for the detection of HPV DNA or RNA in general or HPV 44 DNA orRNA in particular. The labelled precursors which can be used tosynthesize labelled RNA include precursors containing radioactivemarkers such as ³² P, ¹⁴ C, ³ H, ¹²⁵ I or ³⁵ S.

HPV 44 DNA or fragments thereof or HPV 44 RNA or fragments thereof arealso useful as nucleic acid hybridization probes for HPV DNA or RNA ingeneral and HPV 44 DNA or RNA in particular when labelled with anon-radioactive marker such as biotin, an enzyme or fluorescent group.Biotin acts as a hapten-like group and can be bound to the DNA or RNAand detected by binding an avidin-conjugated enzyme orstreptavidin-conjugated enzyme to the biotin followed by washing toremove non-specifically bound enzyme. Upon addition of appropriatesubstrates for the enzyme, the conversion of the substrate to a coloredproduct can be detected (see Leary, J.J. et al, Proc. Natl. Acad. Sci.,USA, 80:4045 (1983)). Examples of such enzymes include alkalinephosphatase and horseradish peroxidase. In addition, fluorescentmolecules such as fluorescein and rhodamine can be chemically conjugatedto avidin or streptavidin and employed as the non-radioactive marker.

Alternatively, the above-described enzymes or fluorescent molecules canbe chemically conjugated directly to the HPV 44 DNA or fragments thereofor HPV 44 RNA or fragments thereof as described in, for example, Renz,M. EMBO J., 6:817 (1983), and used in this manner as hybridizationprobes.

The thus labelled HPV 44 DNA or HPV 44 RNA or fragments thereof can beused as described above in hybridization studies with an unknown sampleof DNA or RNA, particularly an unknown sample of DNA or RNA derived froma genital lesion, to determine if the sample contains HPV DNA or RNA ingeneral and HPV 44 DNA in particular.

The unknown sample of DNA, in addition to being derived from a genitallesion, can be derived from other lesions such as throat, oral or skinlesions.

The unknown sample of DNA or RNA can be obtained by, for example,biopsying an epithelial lesion, scraping the cervix or swabbing thecervix to obtain exfoliated cells. In addition, the unknown sample ofDNA or RNA can be obtained from bacterial cells in which DNA from alesion has been cloned using well known means as described in Maniatis,T. et al, Molecular cloning: A laboratory manual, Cold Spring HarborLaboratory, Cold Spring Harbor, New York (1982) and Gissman, L., CancerSurv., 3:161-181 (1984).

In the methods of the present invention, assaying forcross-hybridization can be carried out by assaying for the presence ofthe radioactive or non-radioactive marker associated withdouble-stranded nucleic acid hybrids. The methods for determiningwhether a specific marker is present will depend upon the markeremployed and are well known in the art.

In the embodiment of the present invention wherein the detection of HPV44 DNA or RNA is based upon a comparison of the epidemiologicaldistribution of cross-hybridization of an unknown sample of DNA or RNAderived from a genital lesion with that of HPV 44 DNA, the unknownsample of DNA or RNA may exhibit less than 50% cross-hybridization withHPV 44 DNA under moderately stringent hybridization conditions, i.e.,using hydroxyapatite chromatography for determining whether two HPVsrepresent different isolates of a common type or represent isolates of adifferent type, yet, may still be considered HPV 44 DNA or RNA by thedefinitions herein. As a result, it is necessary to also compare thecross-hybridization of each lesion which comprises the epidemiologicaldistribution in order to detect HPV 44 DNA or RNA in the sample. This isbecause it may be possible for different HPV types to show the same orsimilar epidemiological distributions. However, by demonstrating thatthe same lesions which comprise the epidemiological distributioncross-hybridize both to HPV 44 DNA and the unknown sample of DNA or RNAderived from a genital lesion it is possible to definitively concludethat the sample of unknown DNA or RNA derived from a genital lesion isHPV 44 DNA or RNA.

HPV 44 DNA or RNA has been found to be present in approximately 1% to 4%of benign cervical lesions but has not been found in any invasivecancers. Thus, HPV 44 DNA or RNA appears to be present in only low gradecervical lesions. On the other hand, other HPV types, such as HPV Types6, 11, 16, 18 and 31 exhibit different distributions in cervical lesionsof various grades. Thus, in the embodiment of the present inventionwherein the detection of HPV 44 DNA or RNA is based upon a comparison ofthe epidemiological distribution of cross-hybridization of an unknownsample of DNA or RNA derived from a genital lesion with that of HPV 44DNA, the unknown sample of DNA or RNA derived from a genital lesionwould cross-hybridize with cervical lesions. If such an epidemiologicaldistribution of cross-hybridization is found with the unknown sample ofDNA or RNA derived from a genital lesion, then this unknown sample ofDNA or RNA may be an HPV 44 DNA or RNA. By demonstrating that the samelesions which comprise the epidemiological distribution alsocross-hybridize with the unknown sample of DNA or RNA derived from agenital lesion, it can be concluded that the unknown sample of DNA orRNA derived from a genital lesion is HPV 44 DNA or RNA.

The particular size of the HPV 44 DNA or HPV 44 RNA fragments which canbe employed as hybridization probes in the present invention is notcritical. The size of the HPV 44 DNA or HPV 44 RNA fragments can be, forexample, from about 15 to about 8000 bases or base pairs, depending onwhether single stranded or double stranded probes are employed,preferably about 300 to about 800 bases or base pairs. When carrying outhybridization in situ, it is preferable that the size of the HPV 44 DNAor HPV 44 RNA fragments is smaller than about 500 bases or base pairssince fragments of this size hybridize in situ more efficiently than HPVDNA or HPV RNA fragments larger than about 1000 bases or base pairs.When using double-stranded DNA or RNA, the DNA or RNA must be denaturedprior to carrying out hybridization.

The HPV 44 DNA fragments can be obtained by restriction endonucleasedigestion of HPV 44 clone 2 or by synthetically manufacturing such usingany of the commerically available DNA synthesizing apparatus or by wellknown chemical methods using the HPV 44 DNA sequence which can bedetermined by well known means (Sanger, S. et al, Proc. Natl. Acad. Sci.USA, 74:5363 (1977)).

When detecting HPV 44 DNA or RNA, it is preferable to use substantiallyall of the HPV 44 genome as a hybridization probe.

The following example is given to further illustrate the presentinvention and is no way intended to limit the scope of the presentinvention. Unless otherwise indicated, all parts, percents, ratios andthe like are by weight.

EXAMPLE (A) Cloning of HPV 44 DNA

The starting material employed was a vulvar condyloma biopsy obtainedfrom Michigan consisting of a few milligrams of tissue. Total DNA waspurified as described in (Maniatis, T. et al, Molecular cloning: Alaboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,New York (1982)). More specifically, the tissue was minced, thendigested in 1.0 ml of 50 mM Tris-HCl, pH 8.0 containing 0.6% (w/v)sodium dodecyl sulfate and 50 μg/ml proteinase K at 37° C. overnight.The resulting digest was extracted twice with 1.0 ml ofphenol:chloroform (1:1 (v/v)). DNA was then precipitated from theaqueous phase by addition of 2 volumes of 90% (v/v) ethanol. Theprecipitated DNA was redissolved in 10 mM Tris, 1.0 mM EDTA buffer, pH8.0 (hereinafter "TE buffer") at a concentration of about 1.0 mg/ml).

The DNA was digested to completion with PstI, electrophased in 1.0%(w/v) agarose gels and DNA transferred to nitrocellulose filters asdescribed in Souther, E.M., J. Mol. Biol., 98:503 (1975). The filterswere then probed under non-stringent hybridization conditions (T_(m)-35° C.) and stringent hybridization conditions (T_(m) -10° C.) with DNAfrom HPV types 6, 11, 16, 18 and 31. Hybridization was performedovernight at 43° C. in 1.0 M NaCl, 50 mM sodium phosphate buffer (pH7.4), 1.0 mM EDTA, 2% (w/v) sodium dodecyl sulfate, O.1% (w/v) gelatin,50 μg/ml tRNA and 30% (v/v) formamide. Four 30 minute washes wereperformed at 55° C. in 1.2X SSC (1X SSC is 0.15 M NaCl plus 0.015 Msodium citrate), 10 mM sodium phosphate (pH 7.4), 1.0 mM EDTA and 0.5%(w/v) sodium dodecyl sulfate. Hybridization was achieved undernon-stringent conditions with HPV types 6, 11, 16, 18 and 31 but notunder stringent hybridization conditions.

The resulting purified DNA was digested with BamHI restrictionendonuclease, which produced a fragment of 7.8 kb (the typical size fora papillomavirus genome). This fragment was cloned into the single BamHIsite of λ L47. More specifically, 2.0 μg of the resulting purified DNA,and 2.0 μg of λ L47 DNA were cut with 10 units of BamHI in a totalvolume of 50 μl of TE buffer for 1 hr at 37° C. The resulting reactionmixture was then diluted with 400 μl of TE buffer and phenol extractedwith an equal volume of phenol:chloroform as described above. Theaqueous phase was then extracted with chloroform:isoamyl alcohol (24:1(v/v)) and DNA from the aqueous phase was precipitated with 80% (v/v)ethanol and dried. The dried DNA was then suspended in 10 μl of 1Xligase buffer comprising 66 mM Tris-HCl, 6.6 mM MgCl₂, 10 mM DTT and 1.0mM ATP and incubated at 42° C. for 2 hours to allow the λ arms toanneal. Next, 0.5 μl of T4 DNA ligase, i.e., about 1 unit, and 0.5 μl of10 mM ATP, pH 7.0 was added to the reaction solution and ligation wasallowed to proceed at 12° C. overnight.

Next, the ligation products were packaged to form infectious phage andused to infect E. coli. NM538. More specifically, a single colony of E.coli NM538 growing on an agarose plate comprising 10 g Tryptone and 5.0g NaCl per liter (hereinafter "TN medium") was selected and grownovernight at 37° C. in 20 ml of TN medium on a shaking platform (250rpm) to early stationary phase. The cell culture was then diluted fourfold with TN medium and grown for 3 hours. Next, the cells wereharvested by centrifuging for 5 minutes at 5,000 rpm in a Sorvall HB-4rotor and the resulting cell pellet was resuspended in 0.25 of theoriginal volume, in 10 mM MgSO₄ and stored at 4° C.

The packaged infectious phages were prepared using a commerciallyavailable BRL Lambda In Vitro Packaging System (see Maniatis, T. et al,Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, New York (1982).

100 μl of an appropriate dilution of packaged phage in phage storagebuffer comprising 0.5 M Tris-HCl (pH 8.0), 0.1 M NaCl, 0.01 M MgSO₄ and0.01% (w/v) gelatin (Difco) to give 1.5×10⁴ plaques per 9 cm diameterplates, was added to 100 μl of E. coli NM538 prepared as described abovein a 10-15 ml test tube, gently mixed and incubated at room temperaturefor 15 minutes. Then, the cell-phage solution was plated on Trypticasesoy broth agar plates comprising 10 g of Trypitcase soy broth, 5.0 gNaCl and 15 g agar per liter, which had been prepared at least one dayin advance and which had been pre-warmed at 37° C. Thereafter, 3-5 ml ofan agarose overlayer comprising 0.5% agarose (ultrapure, electrophoresisgrade) dissolved in 10 mM MgSO₄, which had been heated in a microwaveoven until the solution boiled and then cooled to 45° C. before use, wasplaced over the plated cells-phage. After the agarose had solidified,the plates were transferred to a 37° C. forced air incubator with goodcirculation with the lids of the plates cracked for 30 minutes and thenthe lids were closed and the plates inverted. After 8-12 hours, plaquesbecame apparent.

Infection resulted in confluent lysis of bacteria on the plates.Recombinant phage carrying HPV DNA were localized by performing "plaquelifts" as described by Benton, W.D. et al, Science, 196:180 (1977). Morespecifically, confluent lysed plates were placed at 4° C. for 1 hour toharden the agarose. Then, an appropriately sized piece of nitrocellulosefilter was placed onto each plate by bowing it in the middle, touchingthe center of the plate and working the contact points toward the edge.Then, four assymetric holes were punched through the nitrocellulosefilter and the agar with a small gauge needle and the positions of theholes were marked on the bottom of the plate with a permanent marker.This allowed the nitrocellulose filter and any other areas containingpositive signals to be referenced to corresponding positions on theplates. After 10 minutes, the nitrocellulose filters were removed andthe DNA was denatured by placing the nitrocellulose filters, plaque sidefacing upwards, into a dish containing 200 ml of 0.5 M NaOH, 2.0 M NaClfor 1 minute. The nitrocellulose filters were then neutralized byimmersion in 500 ml of 0.5 M Tris-HCl, 2.0 M NaCl, pH 7.5 for 5 minutes.Next, the filters were rinsed in 6X SSC comprising 0.9 M NaCl, 0.09sodium citrate for 1 minute, dried on Whatman 3 MM paper and then bakedfor 30 minutes at 80° C. under vacuum.

Thereafter, non-stringent hybridization using HPV 16 DNA labelled with³² P by "nick translation" as a probe was carried out on the DNAisolated from the lifted plaques (see Rigby, P.J.W. et al, J. Mol.Biol., 113:237 (1977) and Maniatis, T. et al, Molecular cloning: Alaboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,New York (1982)) followed by washing and autoradiography. Morespecifically, hybridization was performed at 41° C. in a solutioncomprising 1.0 M NaCl, 28% (v/v) formamide, 50 mM N-Tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (hereinafter "TES"),10X Denhardt solution, 0.1 mM EDTA and 10 mM sodium phosphate (pH 7.4).Then a non-stringent wash was carried out at 52° C. using 1.1X SSC(comprising 0.165 M NaCl and 0.0165 M sodium citrate) in 10 mM sodiumphosphate (pH 7.4), 0.1 mM EDTA.

By correspondence with the sites of radioactive exposure, a region ofthe plate containing phage, which contained DNA that hybridized to HPV16 DNA, was excised and used to reinfect E. coli NM538 as describedabove. Localization of phage plaques containing HPV DNA was accomplishedby repeating the above procedure. Six plaques were identified from amongthe 2×10⁵ plaques screened. All clones had identical restriction maps.One of the clones was chosen for further studies and was designated HPV44 clone 1.

The HPV DNA of HPV 44 clone 1 was then digested with BamHI and subclonedin the single BamHI site of pT713. The resulting recombinant DNA wasdesignated HPV 44 clone 2. HPV 44 clone 2 has been deposited at theAmerican Type Culture Collection under ATCC No. 40353.

(B) Characteristic of HPV 44 DNA

1. Hybridization Studies

Hybridization studies were carried out on HPV 44 clone 2 DNA todemonstrate that HPV 44 clone 2 was a new HPV type.

More specifically, ³² P "nick translated" DNA prepared from HPV 44 clone2 was hybridized by Southern blotting under stringent conditions to 5 ngof DNA from HPV Types 1-30, 32-39 and 36-42. DNA from HPV Types 1-30,32-34 and 36-42 were obtained from Dr. Gerard Orth of the InstitutPasteur, Paris, France, the assignor of HPV type designations and Dr.Ethel-Michelle de Villiers of the Papilloma Reference Center inHeidelberg, West Germany. HPV Types 31, 35 and 43 were obtained, fromLife Technologies, Inc. The HPV types were used in pre-immobilized formon nitrocellulose filters. More specifically, hybridization wasperformed at 41° C. in a solution comprising 1.0 M NaCl, 28% (v/v)formamide, 50 mM N-Tris TES, 10X Denhardt solution, 0.5 mM EDTA and 20mM sodium phosphate (pH 7.4). Then, a stringent was was carried out at65° C. using 0.03X SSC (comprising 0.0045 M NaCl and 0.00045 M sodiumcitrate) in 10 mM sodium phosphate (pH 7.4), 0.1 mM EDTA.

While significant homology was detected between the recombinant DNA ofHPV 44 clone 2 and most of HPV types 1 to 43 under non-stringenthybridization conditions, no homology was observed with any of HPV types1-43 under stringent hybridization conditions, thus demonstrating thatHPV 44 clone 2 represents a new HPV type.

2. Restriction Endonuclease Map

The restriction endonuclease map for HPV 44 is shown in FIG. 1. Thefollowing restriction enzymes do not cut HPV 44 DNA: BglI, BglII, ClaI,EcoRV, HindIII, NRUI SalI, SphI, SstI and XhoI.

3. Genomic Organization

In order to demonstrate that the genome of HPV 44 had the same orsimilar open reading frame organization to HPV 6, the followinghybridization studies were carried out. Purified DNA from HPV 44 clone 2was subjected to Southern blotting using ³² P "nick translated"fragments of HPV 6 DNA as a probe under non-stringent conditions andunder stringent conditions as described above. More specifically,fragments of the BamHI-linearized HPV 6b clone were generated with EcoRIand PstI, then gel purified, nick translated with ³² P and used to probeSouthern blots containing NcoI restriction digests of the purified BamHIfragment of HPV 44 clone 2 DNA. The results, which are shown in FIG. 2,demonstrate that the DNA of HPV 44 clone 2 has the same genomicorganization as HPV 6.

4. Epidemiological Distribution

In order to demonstrate that hybridization probes prepared from HPV 44clone 2 hybridizes efficiently under stringent conditions only to HPV 44DNA, and that these hybridization probes can be used to detect genitallesions which contain HPV 44 and to distinguish such genital lesionsfrom genital lesions which contain the DNA of other HPV types, e.g., 6,11, 16, 18, 31 or 33, the DNA of a collection of cervical biopsies andcervical swabs containing exfoliated cells from the Washington, D.C.metropolitan area (including Maryland and Virginia) and Michigan andsurrounding states, were analyzed by nucleic acid hybridization understringent and non-stringent conditions, for the presence of specific HPVDNAs using probes specific for various HPV types, including probesspecific for HPV 44. The biopsies were bisected with half of thespecimens being processed for conventional light microscopy and theother half being frozen and stored at -20° C. for molecular analysis.The tissues on which Southern blot hybridizations were performed weresectioned on a cryostat in order to obtain material for DNA extraction.Approximately every fifteenth section was stained with hematoxylin andeosin stain and examined microscopically in order to confirm that thistissue sample was comparable to the portion of the specimen analyzed bylight microscopy. Exfoliated cervical cells were analyzed by standardcytological methods, e.g., pap smear, on paired samples, the other ofwhich was used for DNA analysis.

High molecular weight DNA was prepared from the samples as describedabove. 1 to 10 μg of purified cellular DNA were digested with eitherPstI or BamHI and the digested samples were electrophoresed in 1.0%(w/v) agarose gels and transferred to nitrocellulose filters.Thereafter, hybridization was carried out under stringent conditions asdescribed above with nick-translated ³² P-labelled HPV DNAs from thetypes discussed above. Note, since the HPV DNAs were propagated in pT713or related vectors, in order to minimize the possibility of reactivitywith pT713 and related vector-like sequences in the tissue samples, allprobes were electrophoretically purified to remove most of theassociated vector sequences. Additional hybridization was also carriedout in the presence of labelled pT713 or pBR322 and related vectors, toreveal any potential false positives due to the plasmid-like sequencesin the tissue samples. The results are shown in Table 1 below. Theresults in Table 1 are graphically illustrated in FIG. 3.

                                      TABLE 1                                     __________________________________________________________________________    Distribution of HPV Types in Cervical Biopsies from the Washington, D.C.      Metropolitan Area and Michigan                                                       Normal                                                                              Metaplastic                                                             Squamous                                                                            Squamous                      Squamous                                                                            Endocervical                        Epithelium.sup.a                                                                    Epithelium.sup.b                                                                    Condyloma                                                                           CIN I CIN II                                                                              CIN III                                                                             Carcinoma                                                                           Adenocarcinoma               __________________________________________________________________________    HPV 6  5     1     17    6     9     1     0     0                                   (1.14%)                                                                             (1.82%)                                                                             (29.31%)                                                                            (11.11%)                                                                            (16.36%)                                                                            (3.57%)                                  HPV 11 0     1     7     2     0     0     0     0                                         (1.82%)                                                                             (12.07%)                                                                            (3.70%)                                              HPV 16 10    1     4     11    22    19    19    2                                   (2.28%)                                                                             (1.82%)                                                                             (6.89%)                                                                             (20.37%)                                                                            (40.0%)                                                                             (67.86%)                                                                            (38.78%)                                                                            (28.57%)                     HPV 18 0     1     1     1     1     2     12    2                                         (1.82%)                                                                             (1.72%)                                                                             (1.85%)                                                                             (1.82%)                                                                              (7.14%)                                                                            (24.40%)                                                                            (28.57%)                     HPV 31 4     0     1     8     7     3     3     0                                   (0.91%)     (1.72%)                                                                             (14.81%)                                                                            (12.73%)                                                                            (10.71%)                                                                            (6.12%)                            HPV 33 3     1     0     3     1     0     1     0                                   (0.68%)                                                                             (1.82%)     (5.56%)                                                                             (1.82%)     (2.04%)                            HPV 44 5     1     2     1     1     0     0     0                                   (1.14%)                                                                             (1.82%)                                                                             (3.44%)                                                                             (1.85%)                                                                             (1.82%)                                        Total No. of                                                                         27    6     32    32    41    25    35    4                            HPV Positive                                                                  Biopsies                                                                      Total No. of                                                                         412   49    27    21    14    3     13    2                            HPV.                                                                          Negative                                                                      Biopsies                                                                      Total No.                                                                            439   55    59    53    55    28    48    6                            of Biopsies                                                                   __________________________________________________________________________     .sup.a These biopsies were from the portio of the cervix.                     .sup.b These biopsies were from the transformation zone.                 

Table 1 and FIG. 3 demonstrate that cervical biopsies containing HPV 44can be distinguished from biopsies containing other HPV types, e.g., 6,11, 16, 18, 31 or 33, not only by the criteria of degree ofcross-hybridization in solution followed by hydroxyapatitechromatography but, also by the ability of an HPV 44 DNA probe tospecifically detect and identify distinct populations of genitallesions, i.e., ones which contain HPV 44 DNA, as compared to ones whichcontain other HPV types. In addition, the results in Table 1 and FIG. 3show that the epidemiological distribution of HPV 44 DNA among cervicalbiopsies is distinct from that found for some other HPV types.

While this invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications could be made therein withoutdeparting from the spirit and scope thereof.

I claim:
 1. A recombinant DNA of HPV 44 comprising a cloning vector andsubstantially all of HPV 44 DNA or fragments thereof.
 2. The recombinantDNA of HPV 44 as claimed in claim 1, wherein said cloning vector isselected from the group consisting of pBR322, pUC11, λ charon, λ L47,M13 derived bacteriophage, pZIP-Neo SV [X1], pBKTK-1, pT712 and pT713.3. The recombinant DNA of HPV 44 as claimed in claim 1, wherein saidfragments are about 15 to about 8000 base pairs in size.
 4. Therecombinant DNA of HPV 44 as claimed in claim 3, wherein said fragmentsare about 300 to about 800 base pairs in size.
 5. The recombinant DNA ofHPV 44 as claimed in claim 1, wherein said recombinant DNA has theidentifying characteristics of HPV 44 clone 2 (ATCC No. 40353).
 6. Therecombinant DNA of HPV 44 as claimed in claim 1, wherein the HPV DNA ofsaid recombinant DNA is labelled with a marker.
 7. The recombinant DNAof HPV 44 as claimed in claim 6, wherein said marker is a radioactivemarker.
 8. The recombinant DNA of HPV 44 as claimed in claim 7, whereinsaid marker is a radioactive marker selected from the group consistingof ³² P, ¹⁴ C, ³ H, ¹²⁵ I and ³⁵ S.
 9. The recombinant DNA of HPV 44 asclaimed in claim 6, wherein said marker is a non-radioactive markerselected from the group consisting of biotin, an enzyme and afluorescent molecule.
 10. The recombinant DNA of HPV 44 as claimed inclaim 9, wherein said enzyme is selected from the group consisting ofalkaline phosphatase and horseradish peroxidase.
 11. The recombinant DNAof HPV 44 as claimed in claim 9, wherein said fluorescent molecule isselected from the group consisting of fluorescein and rhodamine. 12.Essentially pure HPV 44 DNA or fragments thereof.
 13. The essentiallypure HPV 44 DNA as claimed in claim 12, wherein said HPV 44 DNA orfragments thereof are produced biologically.
 14. The essentially pureHPV 44 DNA as claimed in claim 12, wherein said HPV 44 DNA or fragmentsthereof are produced chemically.
 15. The essentially pure HPV 44 DNA asclaimed in claim 12, wherein said fragments are about 15 to about 8000bases or base pairs in size.
 16. The essentially pure HPV 44 DNA asclaimed in claim 15, wherein said fragments are about 300 to about 800bases or base pairs in size.
 17. The essentially pure HPV 44 DNA asclaimed in claim 12, wherein said HPV 44 DNA or fragments thereof arelabelled with a marker.
 18. The essentially pure HPV 44 DNA as claimedin claim 17, wherein said marker is a radioactive marker.
 19. Theessentially pure HPV 44 DNA as claimed in claim 18, wherein said markeris a radioactive marker selected from the group consisting of ³² P, ¹⁴C, ³ H, ¹²⁵ I and ³⁵ S.
 20. The essentially pure HPV 44 DNA as claimedin claim 17, wherein said marker is a non-radioactive member selectedfrom the group consisting of biotin, an enzyme and a fluorescentmolecule.
 21. The essentially pure HPV 44 DNA as claimed in claim 20,wherein said enzyme is selected from the group consisting of alkalinephosphatase and horseradish peroxidase.
 22. The essentially pure HPV 44DNA as claimed in claim 20, wherein said fluorescent molecule isselected from the group consisting of fluorescein and rhodamine. 23.Essentially pure HPV 44 RNA or fragments thereof.
 24. The essentiallypure HPV 44 RNA as claimed in claim 23, wherein said HPV 44 RNA orfragments thereof are produced biologically. ,
 25. The essentially pureHPV 44 RNA as claimed in claim 23, wherein said HPV 44 RNA or fragmentsthereof are produced chemically.
 26. The essentially pure HPV 44 RNA asclaimed in claim 23, wherein said fragments are about 15 to about 8000bases or base pairs in size.
 27. The essentially pure HPV 44 RNA asclaimed in claim 26, wherein said fragments are about 300 to about 800bases or base pairs in size.
 28. The essentially pure HPV 44 RNA asclaimed in claim 23, wherein said HPV 44 RNA or fragments thereof arelabelled with a marker.
 29. The essentially pure HPV 44 RNA as claimedin claim 28, wherein said marker is a radioactive marker.
 30. Theessentially pure HPV 44 RNA as claimed in claim 29, wherein said markeris a radioactive marker selected from the group consisting of ³² P, ¹⁴C, ³ H, ¹²⁵ I and ³⁵ S.
 31. The essentially pure HPV 44 RNA as claimedin claim 28, wherein said marker is a non-radioactive marker selectedfrom the group consisting of biotin, an enzyme and a fluorescentmolecule.
 32. The essentially pure HPV 44 RNA as claimed in claim 31,wherein said enzyme is selected from the group consisting of alkalinephosphatase and horseradish peroxidase.
 33. The essentially pure HPV 44RNA as claimed in claim 31, wherein said fluorescent molecule isselected from the group consisting of fluorescein and rhodamine.
 34. AnHPV 44 hybridization probe comprising a member selected from the groupconsistig of (1) HPV 44 DNA or fragments thereof labelled with a markerand (ii) HPV 44 RNA or fragments thereof labelled with a marker.
 35. TheHPV 44 hybridization probe as claimed in claim 34, wherein saidfragments are about 15 to about 8000 bases or base pairs in size. 36.The HPV 44 hybridization probe as claimed in claim 35, wherein saidfragments are about 300 to about 800 bases or base pairs in size. 37.The HPV 44 hybridization probe as claimed in claim 34, wherein saidmarker is a radioactive member.
 38. The HPV 44 hybridization probe asclaimed in claim 37, wherein said marker is a radioactive memberselected from the group consisting of ³² P, ¹⁴ C, ³ H, ¹²⁵ I and ³⁵ S.39. The HPV 44 hybridization probe as claimed in claim 34, wherein saidmarker is a non-radioactive marker selected from the group consisting ofbiotin, an enzyme and a fluorescent molecule.
 40. The HPV 44hybridization probe as claimed in claim 39, wherein said enzyme isselected from the group consisting of alkaline phosphatase andhorseradish peroxidase.
 41. The HPV 44 hybridization probe as claimed inclaim 39, wherein said fluorescent molecule is selected from the groupconsisting of fluorescein and rhodamine.
 42. An HPV hybridization probecomposition comprising (a) a member selected from the group consistingof (i) HPV 44 DNA or fragments thereof labelled with a marker and (ii)HPV 44 RNA or fragments thereof labelled with a marker and, (b) DNA orRNA or fragments thereof of at least one other HPV type labelled with amarker.
 43. The HPV hybridization probe composition as claimed in claim42, wherein at least any one of said fragments are about 15 to about8000 bases or base pairs in size.
 44. The HPV hybridization probecomposition as claimed in claim 43, wherein at least any one of saidfragments are about 300 to about 800 bases or base pairs in size. 45.The HPV hybridization probe composition as claimed in claim 42, whereinat least any one of said marker is a radioactive member.
 46. The HPVhybridization probe composition as claimed in claim 45, wherein at leastany one of said marker is a radioactive marker selected from the groupconsisting of ³² P, ¹⁴ C, ³ H, ¹²⁵ I and ³⁵ S.
 47. The HPV hybridizationprobe composition as claimed in claim 42, wherein at least any one ofsaid marker is a non-radioactive member selected from the groupconsisting of biotin, an enzyme and a fluorescent molecule.
 48. The HPVhybridization probe composition as claimed in claim 47, wherein saidenzyme is selected from the group consisting of alkaline phosphatase andhorseradish peroxidase.
 49. The HPV hybridization probe composition asclaimed in claim 47, wherein said fluorescent molecule is selected fromthe group consisting of fluorescein and rhodamine.
 50. The HPVhybridization probe composition as claimed in claim 42, wherein saidother HPV type is at least one member selected from the group consistingof HPV 6, HPV 11, HPV 16, HPV 18 and HPV
 31. 51. The HPV hybridizationprobe composition as claimed in claim 50, wherein said other HPV typesis at least one member selected from the group consisting of HPV 16, HPV18 and HPV
 31. 52. The HPV hybridization probe composition as claimed inclaim 51, wherein said other HPV type is HPV 16, HPV 18 and HPV
 31. 53.A method for detecting HPV DNA or RNA comprising:(1) carrying outhybridization, under non-stringent conditions, with (a) a labelled HPV44 nucleic acid selected from the group consisting of (i) HPV 44 DNA orfragments thereof labelled with a marker, and (ii) HPV 44 RNA orfragments thereof labelled with a marker; (b) an unknown sample of DNAor RNA, and (c) at least one other labelled HPV-type nucleic acid; and(2) assaying for the presence of cross-hybridization between alllabelled nucleic acid and unknown sample so as to detect HPV DNA or RNAin said sample.
 54. The method as claimed in claim 53, wherein saidunknown sample of DNA or RNA is derived from a genital, throat, oral orskin lesion.
 55. The method as claimed in claim 54, wherein said unknownsample of DNA or RNA is derived from a genital lesion.
 56. The method asclaimed in claim 55, wherein said unknown sample of DNA or RNA derivedfrom a genital lesion is obtained by biopsying an epithelial lesion,scraping the cervix or by swabbing the cervix to obtain exfoliated cellsor is DNA derived from a genital lesion which has been cloned in acloning vector.
 57. The method as claimed in claim 53, wherein at leastany one of said fragments are about 15 to about 8000 bases or base pairsin size.
 58. The method as claimed in claim 57, wherein at least any oneof said fragments are about 300 to about 800 bases or base pairs insize.
 59. The method as claimed in claim 53, said at least one otherlabelled HPV-type nucleic is selected from the group consisting of:(i)HPV 6 DNA or fragments thereof labelled with a marker; (ii) HPV 6 RNA orfragments thereof labelled with a marker; (iii) HPV 11 DNA or fragmentsthereof labelled with a marker; (iv) HPV 11 RNA or fragments thereoflabelled with a marker; (v) HPV 16 DNA or fragments thereof labelledwith a marker; (vi) HPV 16 RNA or fragments thereof labelled with amarker; (vii) HPV 18 DNA or fragments thereof labelled with a marker;and (viii) HPV 18 RNA or fragments thereof labelled with a marker. 60.The method as claimed in claim 59, wherein said other HPV type is atleast one member selected from the group consisting of HPV 16 and HPV18.
 61. The method as claimed in claim 60, wherein said other HPV typeis HPV 16 and HPV
 18. 62. The method as claimed in claim 53, wherein atleast any one of said marker is a radioactive marker.
 63. The method asclaimed in claim 62, wherein at least any one of said marker is aradioactive member selected from the group consisting of ³² P, ¹⁴ C, ³H, ¹²⁵ I and ³⁵ S.
 64. The method as claimed in claim 53, wherein atleast any one of said marker is a non-radioactive marker selected fromthe group consisting of biotin, an enzyme and a fluorescent molecule.65. The method as claimed in claim 64, wherein said enzyme is selectedfrom the group consisting of alkaline phosphatase and horseradishperoxidase.
 66. The method as claimed in claim 64, wherein saidfluorescent molecule is selected from the group consisting offluorescein and rhodamine.
 67. The method as claimed in claim 53,wherein said cross-hybridization produces DNA-DNA hybrids.
 68. Themethod as claimed in claim 53, wherein said cross-hybridization producesDNA-RNA hybrids.
 69. A method for detecting HPV 44 DNA or RNAcomprising:(1) carrying out hybridization, under stringent conditions,with(a) a member selected from the group consisting of(i) HPV 44 DNA orfragments thereof labelled with a marker, and (ii) HPV 44 RNA orfragments thereof labelled with a marker; (b) an unknown sample of DNAor RNA, and (2) assaying for the presence of cross-hybridization so asto detect HPV 44 DNA or RNA in said sample.
 70. The method as claimed inclaim 69, wherein said unknown sample of DNA or RNA is derived from agenital, throat, oral or skin lesion.
 71. The method as claimed in claim70, wherein said unknown sample of DNA or RNA is derived from a genitallesion.
 72. The method as claimed in claim 71, wherein said unknownsample of DNA or RNA derived from a genital lesion is obtained bybiopsying an epithelial lesion, scraping the cervix or by swabbing thecervix to obtain exfoliated cells or is DNA derived from a genitallesion which has been cloned in a cloning vector.
 73. The method asclaimed in claim 69, wherein said fragments are about 15 to about 8000bases or base pairs in size.
 74. The method as claimed in claim 73,wherein said fragments are about 300 to about 800 bases or base pairs insize.
 75. The method as claimed in claim 69, wherein said HPV 44 DNAcomprises substantially all of the HPV 44 genome.
 76. The method asclaimed in claim 69, wherein said marker is a radioactive member. 77.The method as claimed in claim 76, wherein said marker is a radioactivemarker selected from the group consisting of ³² P, ¹⁴ C, ³ H, ¹²⁵ I and³⁵ S.
 78. The method as claimed in claim 69, wherein said marker is anon-radioactive marker selected from the group consisting of biotin, anenzyme and a fluorescent molecule.
 79. The method as claimed in claim78, wherein said enzyme is selected from the group consisting ofalkaline phosphatase and horseradish peroxidase.
 80. The method asclaimed in claim 78, wherein said fluorescent molecule is selected fromthe group consisting of fluorescein and rhodamine.
 81. The method asclaimed in claim 69, wherein said cross-hybridization produces DNA-DNAhybrids.
 82. The method as claimed in claim 69, wherein saidcross-hybridization produces DNA-RNA hybrids.
 83. A method for detectingHPV 44 DNA or RNA comprising:(1) carrying out hybridization, understringent conditions, with(a) a first fraction of DNA or RNA derivedfrom each individual genital lesion of a sampling of genital lesions,which sampling shows an epidemiological progression to cervical cancer,and (b) a member selected from the group consisting of(i) HPV 44 DNA orfragments thereof labelled with a marker, and (ii) HPV 44 RNA orfragments thereof labelled with a marker; and assaying for the presenceof cross-hybridization with the DNA or RNA derived from each individualgenital lesion or said sampling of genital lesions; (2) carrying outhybridization, under stringent conditions, with(a) a second fraction ofDNA or RNA derived from each individual genital lesion of said samplingof genital lesions, and (b) an unknown sample of DNA or RNA derived froma genital lesion which has been labelled with a marker; and assaying forthe presence of cross-hybridization with the DNA or RNA derived fromeach individual genital lesion of said sampling of genital lesions; (3)comparing the overall patterns of cross-hybridization of said samplingof genital lesions obtaind in Step (1) with that obtained in Step (2);and (4) comparing the cross-hybridizations of each individual genitallesion of said sampling of genital lesions obtained in Step (1) withthat obtained in Step (2);wherein the presence of HPV 44 DNA or RNA insaid unknown sample is detected when both (i) the overall patterns ofcross-hybridization of said sampling of genital lesions are essentiallythe same, and (ii) the cross-hybridizations of each individual genitallesion of said sampling of genital lesions are essentially the same. 84.The method as claimed in claim 83, wherein said fragments are about 15to about 8000 bases or base pairs in size.
 85. The method as claimed inclaim 84, wherein said fragments are about 300 to about 800 bases orbase pairs in size.
 86. The method as claimed in claim 83, wherein saidHPV 44 DNA comprises substantially all of the HPV 44 genome.
 87. Themethod as claimed in claim 83, wherein at least any one of said markeris a radioactive marker.
 88. The method as claimed in claim 87, whereinat least any one of said marker is a radioactive marker selected fromthe group consisting of ³² P, ¹⁴ C, ³ H, ¹²⁵ I and ³⁵ S.
 89. The methodas claimed in claim 83, wherein at least any one of said marker is anon-radioactive marker selected from the group consisting of biotin, anenzyme and a fluorescent molecule.
 90. The method as claimed in claim89, wherein said enzyme is selected from the group consisting ofalkaline phosphatase and horseradish peroxidase.
 91. The method asclaimed in claim 89, wherein said fluorescent molecule is selected fromthe group consisting of fluorescein and rhodamine.
 92. The method asclaimed in claim 83, wherein said unknown sample of DNA or RNA derivedfrom a genital lesion is obtained by biopsying an epithelial lesion,scraping the cervix or by swabbing the cervix to obtain exfoliated cellsor is DNA derived from a genital lesion which has been cloned in acloning vector.
 93. The method as claimed in claim 83, wherein at leastany one of said cross-hybridization produces DNA-DNA hybrids.
 94. Themethod as claimed in claim 83, wherein at least any one of saidcross-hybridization produces DNA-RNA hybrids.